Expression profiles in Caenorhabditis elegans mutants lacking an intestinally expressed microRNA mir-60 that promote adaptive response against chronic oxidative stress

Recent revelations into microRNA function suggest that microRNAs serve as a key player in a robust adaptive response against stress in animals through their fine-tuning capability in gene expression. However, it remains largely unclear how a microRNA-modulated downstream mechanism contributes to the process of homeostatic adaptation. Here we show that loss of an intestinally expressed microRNA gene mir-60 in the nematode C. elegans promotes adaptive response against oxidative stress; animals lacking mir-60 dramatically extend lifespan under a mild and long-term oxidative stress condition, while they do not increase resistance against a strong and transient oxidative stress exposure. We found that canonical stress responsive factors, such as DAF-16/FOXO, are dispensable for mir-60 loss to enhance oxidative stress resistance. Gene expression profiles revealed that genes encoding lysosomal proteases and those involved in the xenobiotic metabolism and pathogen defense response are up-regulated by the mir-60 loss. Detailed genetic studies and computational microRNA target prediction suggest that endocytosis components and a bZip transcription factor gene zip-10, which functions in innate immune response, are directly modulated by miR-60 in the intestine. Our findings suggest that the mir-60 loss facilitates adaptive response against chronic oxidative stress by ensuring the maintenance of cellular homeostasis. Overall design: To identify genes that respond to the mir-60 loss, RNA expression profiles were examined between the mir-60 loss mutant (mir-60(n4947)) and its control animals using the high-throughput sequencing technology. In this study, we used spe-9(hc88), a temperature-sensitive sterile strain, which has been shown in previous studies to have a lifespan similar to wild-type and widely used in gene expression studies to reduce the effect of RNA contamination from younger progenies. Both spe-9 single and mir-60;spe-9 double mutant animals were cultured at a restrictive temperature 23.5 °C, and treated with paraquat 5 mM during adulthood for chronic oxidative stress. Total RNAs were purified at the following time points: Day 0 young adult for both spe-9 and mir-60;spe-9 (just before paraquat exposure); Day 7 for both spe-9 and mir-60;spe-9 (50% survival time for spe-9); Day 10 for mir-60;spe-9 (50% survival time for mir-60;spe-9). For Day 0 controls, total RNAs were isolated twice independently for biological replicates. cDNA libraries were made for these 7 samples with indexed adapters using TruSeq Stranded mRNA Sample Prep Kit (Illumina), and sequenced on 2 lanes of flow cells on the HiSeq 2000/2500 platform, eventually providing 14 sequencing samples.

近期有关微小RNA（microRNA）功能的研究揭示，微小RNA可通过精准调控基因表达，成为动物对抗应激的强效适应性应答中的关键调控因子。然而，微小RNA介导的下游通路如何参与稳态适应过程，目前仍尚不明确。
本研究以秀丽隐杆线虫（C. elegans）为模型，发现肠道表达的微小RNA基因mir-60的缺失可促进机体对抗氧化应激的适应性应答：在温和长期氧化应激条件下，mir-60缺失的线虫寿命显著延长；但该突变体并未增强其对强瞬时氧化应激暴露的抗性。
我们发现，经典应激应答因子如DAF-16/叉头框O（FOXO），对于mir-60缺失介导的抗氧化应激抗性增强并非必需。基因表达谱分析显示，mir-60缺失会上调编码溶酶体蛋白酶的基因，以及参与异生物质代谢和病原体防御应答的基因。深入的遗传学研究与微小RNA靶标计算预测结果表明，肠道内的miR-60可直接调控内吞作用相关组分与参与固有免疫应答的碱性亮氨酸拉链（bZip）转录因子基因zip-10。本研究结果提示，mir-60缺失可通过维持细胞稳态，促进机体对抗慢性氧化应激的适应性应答。

整体实验设计：
为鉴定响应mir-60缺失的基因，本研究采用高通量测序技术，对mir-60缺失突变体（mir-60(n4947)）及其对照动物进行RNA表达谱分析。
本研究使用了温度敏感型不育菌株spe-9(hc88)，既往研究表明该菌株的寿命与野生型相近，且被广泛应用于基因表达研究，以降低年轻子代RNA污染的影响。将spe-9单突变体与mir-60;spe-9双突变体均培养于限制性温度23.5℃，并在成虫期用5mM百草枯处理以施加慢性氧化应激。
分别于以下时间点收集样本并纯化总RNA：第0天的年轻成虫阶段（百草枯处理前），spe-9与mir-60;spe-9两组各设置2次独立生物学重复；第7天，即spe-9单突变体的50%存活时间点，两组均收集样本；第10天，即mir-60;spe-9双突变体的50%存活时间点，仅收集该双突变体样本。上述样本共计7个。
随后，使用Illumina的TruSeq链特异性mRNA样本制备试剂盒，为这7个样本构建带索引接头的cDNA文库，并在HiSeq 2000/2500平台的2个测序流槽上进行测序，最终获得14个有效测序样本。